Project Abstract Toxoplasma gondii is an important human pathogen that causes severe disease in immunocompromised individuals, such as those undergoing chemotherapy, organ transplantation, and AIDS patients. It also afflicts women who become infected for the first time during pregnancy. A healthy immune system or current drug regiment controls the replication of the tachyzoites associated with clinical toxoplasmosis. However, there are no effective therapies to eliminate the chronic stages associated with encysted bradyzoites and, importantly, to prevent the cyst reactivation. To find new avenues for combating the chronic and reactivated toxoplasmosis, we focus on the mechanisms of the tachyzoite and bradyzoite interconversions that are poorly understood. The critical difference between a tachyzoite and a bradyzoite is the rate of parasite replication and the cell cycle architecture. A tachyzoite divides fast and has a relatively short G1 period. In contrast, a bradyzoite rarely divides and spends a progressively longer time in the G1 phase. The time parasite spends in the G1 period is regulated by the RESTRICTION checkpoint (R-point) that in T. gondii lacks conventional regulators. The current application is based on the central hypothesis that the atypical TgCrk2 kinase and P-type cyclins define the novel G1 checkpoint that governs transitions between acute and chronic toxoplasmosis. In our preliminary studies, we showed a differential expression of three P-cyclins in the fast- (RH) and slow-dividing (ME49) T. gondii strains, which also differ in their ability to differentiate. We also showed that all three P-cyclins interact with G1 kinase TgCrk2 in vivo. We believe that P-cyclins differentially regulate levels of TgCrk2 activity, therefore, facilitate or block R-point passage. This dictates the parasite?s choice to either replicate as a tachyzoite or to convert into a resting bradyzoite. To prove our hypothesis, we will define the mechanism of the R-point regulation by TgCrk2 kinase and cyclins TgCycP2, and TgCycP3 in the tachyzoite and bradyzoite development in vitro (Aim 1); in the natural progression of the disease using mouse model (Aim 2); and determine function of the novel R-point components TgCables1 and TgRch1 (Aim 3). Our proposal is built on a strong foundation of advanced genetics, which is bolstered by our extensive experience in studying cyclin/Crk regulators of the T. gondii tachyzoite cell cycle. Using a panel of strains with novel auxin-dependent conditional protein expression of P-cyclins, we will test whether differences in regulation of TgCrk2/P-cyclin complexes explain the dynamics of the bradyzoite differentiation and cyst reactivation. Altogether, our experiments will break new ground in understanding the mechanics and regulation of the developmental switch that regulates the progression of the disease.